Purchase this article with an account.
Gail M Seigel, Kelley Yuan, Zachary K. Goldsmith, Vanessa Marie Morales-Tirado; Heterogeneous R28 retinal precursor cells predominantly express retinal ganglion cell and glial cell markers. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4592.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Over the past twenty years, the R28 retinal cell line has proven to be a valuable model system for the study of retinal cell behavior, differentiation, neuroprotection, cytotoxicity, gene expression and neuronal function. Advanced technologies, coupled with new retinal cell-specific markers have allowed us to learn more about gene expression patterns important for optimal use of this retinal cell line. The purpose of this study was to evaluate the heterogeneity of the R28 cell line using a panel of retinal markers to determine which of the major retinal cell types predominate in R28 cell cultures.
R28 cells were authenticated by STR and species-specific PCR analysis (IDEXX RADIL, Columbia, MO) and confirmed to be of Sprague-Dawley rat origin, with the expected STR expression pattern. We chose 41 genes corresponding to known markers for photoreceptors, amacrine cells, bipolar cells, horizontal cells, astrocytes and Müller cells and optimized probes for qRT-PCR, with HPRT as an endogenous control for comparison purposes. We confirmed PCR analysis by immunocytochemistry and flow cytometry of R28 cell cultures.
Of the glial cell markers tested, we observed very high expression levels of Slc6a9, a GLYT1 glycine transporter found on astrocytes, SLC1a3, a glial high-affinity glutamate transporter, as well as ApoE (involved in lipoprotein metabolism), along with significant levels of vimentin and GFAP. Of the retinal ganglion cell markers tested, we observed (in descending order of expression): Rbpms (an RNA-binding protein highly specific for retinal ganglion cells), Pou4f1 (BRN3A), Tubb3 (Class III beta-tubulin) and Rbfox3. We saw lower levels of expression for genes associated with photoreceptors, horizontal cells and bipolar cells. Nestin and SSEA-1, both stem cell markers, were also expressed by R28 cells.
There is a preponderance of markers for glial cells and retinal ganglion cells in the heterogeneous population of R28 cells. The presence of nestin and SSEA-1 along with these more mature retinal cell markers confirms our early prediction of a precursor cell phenotype with further differentiation potential. A clearer understanding of R28 cell gene expression will help elucidate underlying mechanisms for targeted differentiation into mature retinal cell types.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
R28 cells: nestin (red), DAPI nuclei (blue)
This PDF is available to Subscribers Only